Abstract
The dedifferentiation of chloroplasts in synthetic Brassica napus mesophyll protoplasts in culture was examined. Protoplasts cultured in MS medium containing 0.5 mg/l kinetin and 3 mg/l 2, 4-D were divided frequently and maintained cell viability. This medium was used as a medium of the dedifferentiated system. In the above medium without kinetin, protoplasts were seldom divided and became senile. This medium was used as a medium of the senescent system. Physiological changes of protoplasts were compared between the both systems during culture. Chlorophyll content decreased in both systems. Soluble proteins in whole cells and the chloroplast fraction decreased in the senescent system. In the dedifferentiated system, soluble proteins of whole cells, however, increased and were accompanied by cell division and on the contrary, those of chloroplasts decreased. RNase activity increased in the dedifferentiated system. It increased at first, decreasing in the senescent system afterward. RNase activity in whole cells changed in parallel with that of chloroplasts. However, chloroplast fraction/whole cells in RNase activity gradually became low. SDS-PAGE showed that each chloroplast protein was degraded in both systerns. After 7 days of culture, most of the chloroplast proteins decreased in the senescent system, whereas RuBPCase and CF1 were clearly identified in the dedifferentiated system. Protease activity tended to increase in both systems, and it was stronger in the chloroplast-free fraction than in the chloroplast fraction.
